International Journal of Molecular Sciences

Review Excision of Oxidatively Generated Lesions by Competitive DNA Repair Pathways

Vladimir Shafirovich * and Nicholas E. Geacintov

Chemistry Department, New York University, New York, NY 10003-5180, USA; [email protected] * Correspondence: [email protected]

Abstract: The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. It is generally believed that small non-bulky oxidatively generated DNA base modifi- cations are removed by BER pathways, whereas DNA helix-distorting bulky lesions derived from the attack of chemical or UV irradiation are repaired by the NER machinery. However, existing and growing experimental evidence indicates that oxidatively generated DNA lesions can be repaired by competitive BER and NER pathways in human cell extracts and intact human cells. Here, we focus on the interplay and competition of BER and NER pathways in excising oxidatively generated guanine lesions site-specifically positioned in plasmid DNA templates constructed by a gapped-vector technology. These experiments demonstrate a significant enhancement of the NER yields in covalently closed circular DNA plasmids (relative to the same, but linearized form of the same plasmid) harboring certain oxidatively generated guanine lesions. The interplay between

 the BER and NER pathways that remove oxidatively generated guanine lesions are reviewed and  discussed in terms of competitive binding of the BER proteins and the DNA damage-sensing NER

Citation: Shafirovich, V.; Geacintov, factor XPC-RAD23B to these lesions. N.E. Excision of Oxidatively Generated Guanine Lesions by Keywords: DNA damage; ; nucleotide excision repair; ; reactive Competitive DNA Repair Pathways. oxygen species; guanine oxidation Int. J. Mol. Sci. 2021, 22, 2698. https://doi.org/10.3390/ijms22052698

Academic Editors: Janusz Adam Rak, 1. Introduction Anil Kumar and Endogenous , free radicals and electrophiles generated by UV Magdalena Zdrowowicz light, ionizing radiation and environmental pollutants, are known to induce permanent DNA damage and harmful epigenetic changes [1–3]. It has been estimated that up to 105 Received: 15 February 2021 spontaneous or induced DNA lesions are formed per cell every day [4]. Typical forms Accepted: 4 March 2021 Published: 7 March 2021 of nucleotide modifications, include DNA cross-links, strand breaks, and a variety of oxidatively generated DNA lesions that enhance the rates of and genomic

Publisher’s Note: MDPI stays neutral instability [5,6]. In healthy tissues, genomic stability is maintained by the DNA repair with regard to jurisdictional claims in machinery that removes DNA lesions in an efficient and timely manner [7]. Among the published maps and institutional affil- multiple forms of cellular repair, the base excision repair (BER) pathway generally removes iations. non-bulky oxidatively generated DNA lesions [8]. The mechanisms of BER are highly conserved from bacteria to humans and involve the excision of single DNA lesions [9], while DNA helix-distorting bulky lesions are recognized, excised and repaired by the nucleotide excision repair (NER) mechanism [10]. The structural distortions caused by DNA lesions are recognized by the DNA damage- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. sensing NER factor XPC-RAD23B (abbreviated as XPC) [11]. The initial XPC binding step This article is an open access article constitutes the recognition stage that is followed by the sequential binding of the ten-protein distributed under the terms and factor TFIIH and XPA, XPF and XPG proteins to the site of the lesion. The hallmark of conditions of the Creative Commons the NER excision mechanism is the appearance of the characteristic ~24–30 nucleotide (nt) Attribution (CC BY) license (https:// dual incision products that contain the lesion [10]. Deficiency in XPC levels is associated creativecommons.org/licenses/by/ not only with decreased rates of repair of certain oxidatively generated DNA lesions, but 4.0/). also to compromised redox homeostasis and loss of cell cycle control [12,13].

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Growing evidence suggests that the BER and NER pathways can compete with one another in removing oxidatively generated lesions from double-stranded DNA [14–17]. In this contribution, we summarize more recent experimental results that provide further insights into the competition between the BER and NER pathways in excising hydantoin lesions that are generated by the oxidation of guanine [14,18–21]. A significant enhance- ment (by factor of 5–6) of the NER dual incision yields of hydantoin lesions embedded in covalently closed circular DNA plasmids (cccDNA) than in the linearized form of the same plasmid DNA (linDNA) has been observed [21,22]. By contrast, the BER yields in cccDNA and linDNA did not vary by more than 20–40%, depending on the guanine lesion. These surprising differences in NER and BER activities have been attributed to the lack of termini in covalently closed circular DNA, that enhance the search dynamics of the NER DNA damage sensor XPC in circular DNA plasmid molecules.

2. Guanine Lesions Generated by Electron Abstraction and Free Oxidation Pathways Guanine, the most easily oxidizable base [23], is a primary target of oxidizing agents [24]. Among the four canonical DNA bases, guanine has the lowest redox potential (E7 = 1.26 V vs. NHE [23]), and is readily oxidized by carbonate radical anions •- 0 (CO3 ), a mild oxidizing agent (E = 1.59 V vs. NHE [25]). The carbonate radical anion is generated in cellular environment by the decomposition of nitrosoperoxycarbonate (ONOOCO2)[2]. Oxidation of other nucleobases (A, C, and T) [26–28] requires significantly •− 0 stronger oxidants, such as the sulfate radical, SO4 (E = 2.43 V vs. NHE [25]. Guanine oxidation is typically initiated either by one-electron abstraction or by hy- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 12 droxyl radical addition reactions (Figure1A) [15,26].

Figure 1. (A) Oxidatively generated guanine lesions generated by reactions of strong oxidants or Figure 1. (A) Oxidatively generatedelectrophilic guanine free lesions radicals. 8-Oxo-7,8-dehydr generatedoguanine by reactions (8-oxoG, via of C8 strongaddition of oxidantsH2O/•OH), and or electrophilic free radicals. 5-carboxamido-5-formamido-2-iminohydan•toin (2Ih, by C5 addition of H2O/•OH), whereas end 8-Oxo-7,8-dehydroguanine (8-oxoG,products via C8 of a four-electron addition oxidation of H2 areO/ spiroiOH),minodihydantoin and 5-carboxamido-5-formamido-2-iminohydantoin (Sp) and 5-guanidinohydantoin • (Gh). The two-electron oxidation products are substrates of BER only [29,30], while the four-elec- (2Ih, by C5 addition of H2O/ OH), whereastron oxidation endproducts products are substrates of of both a four-electron BER and NER pathways oxidation [14,21,31]. (B are) Thespiroiminodihydantoin bulky (Sp) and 5-guanidinohydantoin (Gh). The two-electronguanine DNA adduct oxidation 10R-(+)-cis-anti products-B[a]PDE-N are2-dG substrates is a well-known substrate of BER of NER only only [29 ,30], while the four-electron [32,33] and is used for comparing the NER yields of oxidative DNA lesions that are substrates of oxidation products are substrates ofNER both and BER. BER and NER pathways [14,21,31]. (B) The bulky guanine DNA adduct 10R- 2 (+)-cis-anti-B[a]PDE-N -dG is a well-knownThe free radical substrate intermediates, of NER the guanine only radical [32,33 cation] and (G•+ is) and used the neutral for comparing gua- the NER yields of nine radical [G(-H)•] are highly reactive, and are rapidly hydrated via the addition of H2O oxidative DNA lesions that are substratesmolecules of to either NER the and C5 or BER. C8 position of guanine [34,35]. The 8-HO-G• and 5-HO-G• radicals formed, which are identical to the radical adducts derived from the addition of hydroxyl radicals to the C8 and C5 positions, are reducing agents. These intermediates are rapidly oxidized by O2 or other relatively weak oxidants, to form two stable end-prod- ucts of two-electron oxidation, 8-oxo-7,8-dehydroguanine (8-oxoG) and 5-carboxamido-5-

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The free radical intermediates, the guanine radical cation (G•+) and the neutral gua- nine radical [G(-H)•] are highly reactive, and are rapidly hydrated via the addition of • H2O molecules to either the C5 or C8 position of guanine [34,35]. The 8-HO-G and 5-HO-G• radicals formed, which are identical to the radical adducts derived from the addition of hydroxyl radicals to the C8 and C5 positions, are reducing agents. These intermediates are rapidly oxidized by O2 or other relatively weak oxidants, to form two stable end-products of two-electron oxidation, 8-oxo-7,8-dehydroguanine (8-oxoG) and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) [34–38]. The latter exists in the form of two diastereomers, R-2Ih and S-2Ih, which are chemically stable and can be individ- ually isolated by HPLC methods [34,39]. Alshykhly et al. [30] reported that both 2Ih diastereomers are typical BER substrates and can be removed from damaged DNA by the glycosylases NEIL1 and Fpg. It has been demonstrated that unrepaired, oxidatively generated DNA lesions are associated with germline mutations in tumor suppressor and proto-oncogenes that lead to adenoma-colorectal cancers [40]. The widely studied 8-oxoG product is one of the most abundant and best characterized oxidatively generated DNA lesion, and is a classical BER substrate that is formed in cellular environments under conditions of ox- idative stress [21,41,42]. The 8-oxoG lesions are genotoxic, and failure to remove 8-oxoG before replication occurs, results in the formation of G:C→T:A mutations [43]. Furthermore, 8-oxoG is more easily oxidized than the parent guanine [44], and reacts with •− • •− • diverse oxyl radicals (CO3 , NO2, SO4 , RO )[34,45–50], and peroxynitrite [51,52] to yield the stable end-products of four-electron oxidation of guanine, spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) [53–59]. Due to the presence of chiral carbon atoms, Sp and Gh exist in two diastereoisomeric S and R forms. The Sp-modified oligodeoxynu- cleotides can be separated and purified by anion-exchange HPLC methods [60,61]. In contrast, the Gh diastereomers are easily interconvertible, and can isomerize to iminoal- lantoin (Ia) (Figure1A) [ 54]. In DNA, isomerization of Gh to Ia occurs in basic solutions (pH > 8.2) [62]. The Sp and Gh lesions have been detected in mice with infection-induced colitis, at concentration levels of about one percent, relative to the more abundant 8-oxoG levels [63]. The hydantoin lesions are at least one order of magnitude more mutagenic than the parent 8-oxoG (mainly via G→C and G→T transversion mutations) [64]). The hydantoin lesions are efficiently repaired by BER that include the prokaryotic E. coli Fpg [65] and Nei [66], the mammalian NEIL1 and NEIL2 [67], NEIL3 [68–72], and the human NEIL1 [31,73] and NEIL3 [72] DNA glycosylases. These lesions are substrates of prokaryotic BER and NER mechanisms [74], in cell extracts from HeLa cells and human fibroblasts [14,21], as well as in intact HeLa cells and human fibroblasts [19]. As a positive control of NER activity, we employed the bulky 10R-(+)-cis-anti-B[a]PDE-N2-dG adduct (ab- breviated as B[a]P-dG), an excellent substrate of the human NER pathway [14,19,22,32,33].

3. Construction of Plasmid Substrates Harboring Single Guanine Lesions by a Gapped-Vector Technology Recently we demonstrated that the interplay of BER and NER pathways in cova- lently closed circular plasmids dramatically differs from that in the same, but linearized plasmids [21,22]. A gapped-vector technology [75–80] was employed for the site-specific insertion of single guanine lesions into pUC19NN plasmid molecules (contour length of 2686 bp). The parent pUC19NN plasmid was cloned from the well-known pUC19 plas- mid by inserting a 32-mer 20-deoxyoligonucleotide fragment containing two Nt. BbvCI restriction sites separated by 21 nucleotides (Figure2). Int. J. Mol. Sci. 2021, 22, 2698 4 of 11 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 12

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 12

Figure 2. 2. CovalentlyCovalently closed closed circular circular and linearized and linearized plasmid plasmid (2686 bp) (2686 containing bp) containing site-specifically site-specifically 32 32 positionedpositioned guanine guanine lesions lesions (X) (X) and and a radioactive a radioactive P-internalP-internal label. The label. circ Theular plasmid circular substrates plasmid substrates werewere generated by by gapped-vector gapped-vector technology technology fromfrom a pUC19NN a pUC19NN plasmid plasmid containing containing two Nt. BbvCI two Nt. BbvCI restrictionFigure 2. Covalently sites [21,22]. closed The linearizedcircular and plasmid linearized subs pltrateasmid with (2686 the lesionbp) containing X positioned site-specifically at the 945th 32 restrictionnucleotidepositioned countedguanine sites [21 fromlesions,22]. the The (X) 5′ -end,and linearized a was radioactive prepared plasmid P-internalby substratethe selective label. with cleavageThe the circ lesionular of the plasmid Xcircular positioned substrates plasmid at the 945th 0 nucleotidewithwere agenerated unique counted ScaI by restrictiongapped-vector from the 5 -end,technology th wasat generates preparedfrom a bluntpUC19NN by end the cleavageselective plasmid products. containing cleavage oftwo the Nt. circular BbvCI plasmid withrestriction a unique sites ScaI[21,22]. restriction The linearized enzyme plasmid that generatessubstrate with blunt the end lesion cleavage X positioned products. at the 945th nucleotideThe gapped counted plasmidfrom the 5derived′-end, was from prepared the nickin by theg selectiveof pUC19NN cleavage with of the the circular Nt. BbvCI plasmid re- with a unique ScaI restriction enzyme that generates blunt end cleavage products. strictionThe enzyme, gapped was plasmid filled derivedwith the from5′-phosphorylated the nicking ofoligonucleotides pUC19NN with 5′-pTCAGCGA- the Nt. BbvCI restric- TAT and 32P-endlabeled 5′-32pCCATCXCTACC0 (where the lesion X = Gh, S-Sp,0 8-oxoG, or tion enzyme,The gapped was plasmid filled withderived the from 5 -phosphorylated the nicking of pUC19NN oligonucleotides with the Nt. 5 -pTCAGCGATATBbvCI re- cis-B[32a]P-dG), and was ligated0 32 to the plasmid by T4 ligase. The reaction products were andstrictionP-endlabeled enzyme, was filled 5 - pCCATCXCTACCwith the 5′-phosphorylated (where oligonucleotides the lesion X =5′-pTCAGCGA- Gh, S-Sp, 8-oxoG, or treated with T5 exonuclease to digest any linear and nicked plasmids [79–81]. The cleav- cisTAT-B[ anda]P-dG), 32P-endlabeled and was 5′- ligated32pCCATCXCTACC to the plasmid (where by the T4 lesion ligase. X = The Gh, reactionS-Sp, 8-oxoG, products or were age of the covalently closed circular plasmid substrates by the unique restriction enzyme treatedcis-B[a]P-dG), with T5and exonuclease was ligated toto digest the plasmid any linear by T4 and ligase. nicked The plasmidsreaction products [79–81]. were The cleavage ScaI generates linearized plasmids with blunt ends with a guanine lesions (X) at the 945th oftreated the covalently with T5 exonuclease closed circular to digest plasmid any linear substrates and nicked by theplasmids unique [79–81]. restriction The cleav- enzyme ScaI nucleotide counted from the 5′-end. generatesage of the covalently linearized closed plasmids circular with plasmid blunt substrates ends with by the a guanineunique restriction lesions (X)enzyme at the 945th ScaI generates linearized plasmids0 with blunt ends with a guanine lesions (X) at the 945th nucleotide4. Monitoring counted Competing from BER the 5and-end. NER Pathways with Single DNA Lesions Embed- nucleotide counted from the 5′-end. ded in Plasmids 4. Monitoring Competing BER and NER Pathways with Single DNA Lesions Embedded4. MonitoringThe formation in Competing Plasmids of NER BER and and BER NER excision Pathways products with was Single monitored DNA Lesions by 32P-internally Embed- labelledded in Plasmids plasmid substrates harboring single lesions, by high resolution denaturing32 poly- acrylamideThe formation gel electrophoresis of NER methods and BER [21,22]. excision After products incubation was of the monitored plasmid substrates by P-internally The formation of NER and BER excision products was monitored by 32P-internally labelledin cell extracts, plasmid the substrates DNA samples harboring were isolated single lesions,and treated by highwith resolutionEcoRI and denaturingBsrBI re- poly- labelled plasmid substrates harboring single lesions, by high resolution denaturing poly- acrylamidestriction enzymes gel electrophoresis to excise 32P-labeled methods 40-mer [21 fragments,,22]. After the incubation products of thesuccessful plasmid BER substrates acrylamide gel electrophoresis methods [21,22]. After incubation of the plasmid substrates inactivity, cell extracts, or the unincised the DNA and samples intact 10 were1-mer isolated fragments and as treated shown with in Figure EcoRI 3. and BsrBI restriction in cell extracts, the DNA samples were isolated and treated with EcoRI and BsrBI re- enzymes to excise 32P-labeled 40-mer fragments, the products of successful BER activity, or striction enzymes to excise 32P-labeled 40-mer fragments, the products of successful BER theactivity, unincised or the unincised and intact and 101-mer intact 10 fragments1-mer fragments as shown as shown in Figure in Figure3. 3.

Figure 3. Schematic summary of the analysis of products derived from the incisions of covalently closed circular plasmids by BER and NER mechanisms after incubation in human cell extracts [21,22]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 12

Figure 3. Schematic summary of the analysis of products derived from the incisions of covalently closed circular plasmids by BER and NER mechanisms after incubation in human cell extracts [21,22].

Successful NER activity generates the dual incision products ~24–32 nucleotides in lengths containing the 32P-label, as well as the lesion [82,83]. A similar approach was em- ployed to determine the yields of BER and NER excision products using linDNA contain- Int. J. Mol. Sci. 2021, 22, 2698 ing single Sp and Gh lesions [21,22]. 5 of 11

5. Remarkable Enhancement of NER of Guanine Lesions in Covalently Closed Circu- lar SuccessfulPlasmids NER Relative activity to generates the Same, the dualBut Linearized incision products Plasmids ~24–32 nucleotides in lengths containing the 32P-label, as well as the lesion [82,83]. A similar approach was employedWe torecently determine discovered the yields that of BER the and NER NER dual excision incision products yields using are linDNAenhanced by factor of containing5–6 when single guanine Sp and lesions Gh lesions are [embedded21,22]. in covalently closed circular pUC19NN plasmids rather than in the same, but linearized plasmid containing single Sp, Gh lesions or (+)-cis- 5. Remarkable Enhancement of NER of Guanine Lesions in Covalently Closed CircularB[a]P-dG Plasmids adducts Relative by treatment to the Same, of Butthe Linearized circular pUC19NN Plasmids plasmid with the restriction en- zymeWe ScaI recently (Figure discovered 2) [21,22]. that the In NERthese dual experi incisionments, yields we are used enhanced the hydantoin by factor of lesions (Gh and 5–6S-Sp), when which guanine are lesions substrates are embedded of both in covalentlyBER andclosed NER circularpathways, pUC19NN and employing plasmids 8-oxoG as a ratherpositive than controls in the same, of the but BER linearized and NER plasmid pathways, containing respectively. single Sp, Gh lesions or (+)- cis-B[a]P-dGThe denaturing adducts by treatment polyacrylamide of the circular gel pUC19NNelectropho plasmidresis clearly with the show restriction that incubation of enzyme ScaI (Figure2)[ 21,22]. In these experiments, we used the hydantoin lesions (Gh andtheS S-Sp),-Sp-plasmids which are substrates in HeLa of bothcell BERextracts and NER follow pathways,ed by and treatment employing with 8-oxoG EcoRI as and BsrBI re- astriction positive controls enzymes of the generates BER and NERthree pathways, groups respectively.of 32P-labeled DNA fragments: (1) the character- isticThe ladders denaturing of NER polyacrylamide dual incision gel electrophoresis products of clearly ~20–32 show nucleotides that incubation in of lengths, the (2) the 40- Smer-Sp-plasmids fragments in HeLa generated cell extracts by followed BER activity, by treatment and with (3) EcoRIthe 101-mer and BsrBI fragment restriction excised by the enzymes generates three groups of 32P-labeled DNA fragments: (1) the characteristic restriction enzymes from the unincised and intact plasmids (Figure 3) [21]. A similar re- ladders of NER dual incision products of ~20–32 nucleotides in lengths, (2) the 40-mer fragmentsmarkable generated enhancement by BER activity, of the andNER (3) dual the 101-mer incision fragment yields excised was byalso the evident restriction in the case of Gh enzymeslesions. fromThe the8-oxoG unincised lesions and in intact the same plasmids plasmi (Figureds3 are)[ 21 exclusively]. A similar repaired remarkable by the BER path- enhancementway [21]. In of turn, the NER the dual (+)- incisioncis-B[a yields]P-dG was adduct also evident is removed in the case by of the Gh lesions.NER mechanism The only, as 8-oxoGexpected lesions [22]. in the same plasmids are exclusively repaired by the BER pathway [21]. In turn, the (+)-cis-B[a]P-dG adduct is removed by the NER mechanism only, as expected [22]. TheThe effects effects of plasmid of plasmid linearization linearization on the ratio on of the the ra relativetio of yieldsthe relative of BER andyields NER of BER and NER incisionincision products products (Yccc DNA(Yccc/YDNAlin/YDNAlinDNA)[21), 22[21,22]] are summarized are summarized in Figure in4. Figure 4.

FigureFigure 4. 4.Impact Impact of of plasmid plasmid linearization linearization on the on ratios the ofratios the relative of the relative yields of yields BER and of NERBER and NER inci- incision products (Y /Y ) after incubation of 32P-internally labelled plasmids harboring sion products (YccccccDNADNA/YlinlinDNADNA) after incubation of 32P-internally labelled plasmids harboring S-Sp, SGh,-Sp, 8-oxoG, Gh, 8-oxoG, and and (+)- (+)-ciscis-B[-B[a]P-dGa]P-dG lesions lesions inin HeLaHeLa cell cell extracts. extracts. n.d.–not n.d. – detected.not detected. (Data (Data from Kol- from Kolbanovskiy et al. Biochemistry, 2020, 59, 2842–2848 [21], and Chem. Res. Toxicol., 2021, 34, banovskiy et al. Biochemistry, 2020, 59, 2842–2848 [21], and Chem. Res. Toxicol., 2021, 34, 154–160 154–160 [22]). n.d.–not detected. [22]). n.d. – not detected.

This figure shows that the (YcccDNA/YlinDNA) ratios are 4.8 ± 0.5 (Sp), 5.1 ± 0.5 (Gh) [21], and 6.0This± 0.5 figure (B[a]P-dG) shows [20 ],that thus the indicating (YcccDNA that/Ylin theDNA NER) ratios activities are 4.8 of both± 0.5 hydantoin (Sp), 5.1 ± 0.5 (Gh) [21], lesionsand 6.0 and ± B[ 0.5a]P-dG (B[a adduct]P-dG) in circular[20], thus plasmids indicating are greater thatthan the in linearizedNER activities plasmids of by both hydantoin a factor of ~5–6. However, the relative BER yields of the Sp and Gh lesions are reduced by

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~20–30% in circular plasmids. This decrease in BER activities of Sp and Gh in circular plas- mids might be due to the competition between XPC and the BER enzyme for binding to the Sp or Gh lesions [20] since the NER pathway is highly favored in circular DNA molecules. However, a somewhat greater BER activity by ~30–40% in cccDNA is observed in the case of 8-oxo-G in circular plasmids. The remarkable enhancement in NER yields in circular rel- ative to linearized plasmid DNA molecules, suggests the hypothesis that this enhancement is associated with an effectively greater level of bound XPC molecules in circular DNA than in the linearized form. Since the contour lengths and base sequence context are identical in both cases, the smaller NER yield in the linear plasmid molecules is attributed to the two DNA termini that are absent in circular DNA. Single molecule experiments have shown recently that XPC diffuses along linear DNA molecules by a combined sliding and hopping mechanism [84]. The probability of sliding or hopping off the DNA molecule at the ends in linearized DNA molecules is enhanced, thus diminishing the probability that the same XPC molecule will locate the DNA lesion [22]. Indeed, Mason et al. [85] demonstrated earlier that NER excision efficiencies are significantly enhanced in human cell extracts in 149-mer DNA duplexes when the ends are blocked by streptavidin-biotin complexes. Thus, the XPC molecules were prevented from dissociating at both ends of the DNA molecules thus enhancing the observed NER yields. End-effects may play an important role in , an important factor in intracellular DNA repair [86–89]. Whitehouse et al. demonstrated that the ATP-dependent displacement of histone octamer cores induced by SWI/SNF chromatin remodeling complexes was blocked by streptavidin-coated magnetic beads [90].

6. Competition of BER and NER Pathways in Repair of Oxidatively Generated Guanine Lesions The interplay of BER and NER in the repair of hydantoin lesions can be explained by a competitive binding of BER and NER proteins to the Sp and Gh lesions [15]. In principle, the ratios of incision products generated at a given concentration of DNA substrates could be limited by the relative concentrations of one or the other kind of DNA repair protein. Such effects could manifest themselves at different DNA substrate concentrations at constant BER and NER protein concentrations. Indeed, we have recently found that a five-fold rise of DNA concentrations from 0.2 nM to 1 nM induces a decrease in the NER/BER yield ratios from ~4.3 to ~2.7 and ~2.0 to ~1.2 in the case of Sp- and Gh-plasmids, respectively [21]. These results indicate that the observed small decreases in NER/BER ratios as a function of DNA substrate concentration, are due to limiting concentrations of NER proteins in cell extracts. The enhancement of BER yields induced by the addition of DNA glycosylase NEIL1 is correlated with the suppression of the yields of NER dual incision products in cell extracts; this observation indicates that the NER/BER ratios are determined by a competition between NEIL1 and the initial NER DNA lesion recognition factor XPC [14]. Recent experiments with purified human NEIL1 and XPC proteins provide direct support for the competitive binding model of these proteins to the hydantoin lesions site-specifically positioned in 147 bp linear duplexes [20]. Monitoring the glycosylase/lyase activity of the bifunctional DNA glycosylase NEIL1, we have shown that the DNA damage-sensing NER factor XPC reduces the rates of incisions of hydantoin Gh or Sp lesions embedded in double-stranded DNA. Numerical analysis of the kinetic data indicates that both NEIL1 and XPC proteins bind rapidly to Gh or Sp substrates with rate constants close to the diffusion limit for bimolecular association rate constants of other proteins [91,92]. Thus, the preliminary partitioning of binding of NEIL1 and XPC to Gh/SpDNA hydantoin DNA lesions is determined by free diffusion mechanisms. At cellular levels, similar competitive processes between the NER XPC and BER NEIL1 proteins may play a role in the 5–10 times more efficient repair of hydantoin lesions by BER than by NER mechanisms in intact cells [19]. In human mesothelial cells, the nuclear concentrations of NEIL1 are in the range of 250–800 nM [93], while the concentrations of XPC in human fibroblasts were reported to be 140 nM [94]. Int. J. Mol. Sci. 2021, 22, 2698 7 of 11

7. Concluding Remarks and Future Outlook The major pathways of repair of DNA lesions include the base excision repair (BER) mechanism that excises small non-bulky, oxidatively generated DNA lesions, and nu- cleotide excision repair (NER), a mechanism that removes a large spectrum of mostly bulky DNA adducts generated by UV irradiation or environmental pollutants. A small number of non-bulky, oxidatively generated lesions such as the 5,8-cyclopurine lesions [95,96] are exclusive substrates of NER. However, the oxidatively generated hydantoin lesions (Gh, and Sp), derived from the oxidation of 8-oxoguanine, are substrates of both NER and BER [14,19–21]. The interplay between these major repair pathways opened the possibility of comparing the effects of DNA length and topological constraints due the cyclization of DNA, on the relative efficiencies of repair by these two repair pathways. The slower NER efficiencies in the case of linearized forms of the plasmids are correlated with the dissociation of the NER DNA lesion recognition factor XPC from the ends of the linearized plasmid that diminishes the NER efficiency. By contrast, cyclization diminishes the BER yields of the same lesions by ~10–20%. We proposed the hypothesis that these effects can be attributed to the differences in the lesion search mechanisms of BER and NER proteins, that deserve to be investigated in detail. Future studies of the effects of chromatinization of circular instead of linearized plasmid substrates may provide new insights into the poorly understood mechanisms of DNA repair in chromatin contexts that requires the removal of histone core particles by chromatin remodeling factors [86–89]. The relationship between different pathways of DNA repair could provide a better understanding of the etiology of various human diseases that are associated with oxidatively generated DNA damage and the inflammatory response.

Funding: This work was supported by the National Institute of Environmental Health Sciences grant R01 ES-027059 to V.S. and by R21 ES-028546 to N.E.G. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

BER base excision repair; NER nucleotide excision repair; 8-oxoG 8-oxo-7,8-dihydroguanine; 2Ih 5-carboxamido-5-formamido-2-iminohydantoin; Sp spiroiminodihydantoin; Gh 5-guanidinohydantoin; Ia iminoallantoin; B[a]P-dG 10R-(+)-cis-anti-B[a]PDE-N2-dG adduct; G•+ guanine radical cation; G(-H)• guanine neutral radical; bp base pair.

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